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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:954-962.)
© 1997 American Heart Association, Inc.

Articles

Expression of the PAF Receptor in Human Monocyte–Derived Macrophages Is Downregulated by Oxidized LDL

Relevance to the Inflammatory Phase of Atherogenesis

Dominique Stengel; Micheline Antonucci; Muriel Arborati; Delphine Hourton; Sabine Griglio; M. John Chapman; ; Ewa Ninio

From INSERM Unité 321, Unité de Recherche sur les Lipoprotéines et l'Athérogénèse, Pavillon Benjamin Delessert, Hôpital de la Pitié, Paris, France.

Correspondence to Dr Dominique Stengel, INSERM Unité 321, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France. E-mail stengel{at}infobiogen.fr

	Abstract

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Abstract Human monocyte–derived macrophages play a major role in the initiation and progression of atherosclerotic lesions as a result of the production of a wide spectrum of proinflammatory and prothrombotic factors. Among such factors is a potent inflammatory phospholipid, platelet-activating factor (PAF), which is produced after macrophage activation. Because the cells involved in PAF biosynthesis are typically targets for the bioactions of PAF via specific cell surface receptors, we evaluated the expression of the PAF receptor in human monocyte–derived macrophages. Oxidized LDL (oxLDL) exerts multiple cellular effects that enhance lesion progression; we therefore investigated the potential modulation of expression of the macrophage PAF receptor by oxLDL. [³H]PAF bound to adherent human macrophages with a K_d of 2.1 nmol/L and a B_max of 19 fmol/10⁶ cells; 5300 binding sites per cell were detected. OxLDL (100 µg protein per milliliter) induced a twofold decrease in cellular PAF binding after 3 hours at 37°C. Analysis of macrophage mRNA by reverse transcription–polymerase chain reaction (RT-PCR) revealed two forms corresponding to the PAF receptor, of which the leukocyte type (type 1 promoter) predominated. Expression of PAF receptor mRNA, evaluated by quantitative RT-PCR using an actin or a GAPDH mimic, was progressively reduced (up to 70%) by oxLDL up to 6 hours and remained low for at least 24 hours. Such downregulation was reversible after incubation of the cells for 24 hours in oxLDL-free medium. Addition of forskolin (3 µmol/L) or dibutyryl cAMP (1 mmol/L) to macrophage cultures reproduced the oxLDL-mediated inhibition of PAF receptor expression; carbamyl PAF reduced PAF binding and PAF mRNA to a similar degree (50%). These data demonstrate that atherogenic oxLDL downregulates the expression of both cellular PAF receptors and PAF receptor mRNA in macrophages, consistent with both a diminished bioresponse to PAF and decreased cell motility. Such diminished bioresponse to a powerful antacoid reflects the suppression of an acute inflammatory reaction, thereby leading to chronic, low-level inflammation, such as that characteristic of fatty streaks and more advanced atherosclerotic plaques.

Key Words: platelet-activating factor • scavenger receptor • lipid peroxidation • adenylyl cyclase • cAMP • forskolin

	Introduction

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It is now established that human monocyte–derived macrophages and foam cells play a key role in the formation of atherosclerotic plaques.¹ These highly active cells synthesize a myriad of proinflammatory and prothrombotic substances, including PAF-acether, a potent phospholipid mediator of inflammation, thrombosis, and anaphylaxis.^{2 3} Equally, cells involved in PAF biosynthesis are targets for the bioactions of PAF, whose effects are mediated via a specific cell-surface receptor that has been cloned from guinea pig lung⁴ and human leukocytes.^{5 6}

The PAF receptor is a member of the 7-transmembrane-domain receptor family coupled to G proteins⁵ and transduces extracellular signals to intracellular effectors, such as the calcium mobilization system and enzymes involved in phosphoinositide turnover⁵ and protein phosphorylation.⁷ The presence of mRNA for the PAF receptor has been detected in several tissues, including the brain, kidney, spleen, liver, lung, and heart,⁴ and in circulating cells, such as monocytes, neutrophils, and the EoL-1 cell line.⁵ Expression of the PAF receptor may be regulated by two distinct promoters.⁸ Indeed, PAF receptor mRNA from EoL-1 cells, leukocytes, and the brain is expressed only from promoter 1, whereas spleen, kidney, and heart PAF receptor mRNAs are expressed from promoters 1 and 2, thereby indicating potential variability in gene regulation in different tissues. Recently, Ali et al⁹ showed that PAF is able to stimulate phosphorylation of its own receptor in a manner similar to phorbol 12-myristate 13-acetate in a rat basophilic cell line stably transfected with the PAF receptor. Furthermore, a single PAF receptor interacts with multiple G proteins to mediate its biological responses,⁹ whereas its expression may be downregulated by agents that stimulate cAMP formation, such as prostaglandin E₂ and forskolin.¹⁰

A key feature of atherogenesis involves the entry of both LDL and monocytes into the arterial intima. Monocytes mature into tissue macrophages in the subendothelial space and acquire the ability to recognize and internalize various forms of oxLDL via multiple receptors, including scavenger,¹¹ CD36,¹² and Fc¹³ receptors; such uptake leads to intracellular cholesterol accumulation and foam cell formation. Macrophage-derived foam cells are characteristic of both early and advanced atheromatous lesions and, like macrophages, may undergo phagocyte- and cytokine-mediated activation in arterial tissue, thereby contributing to the inflammatory reaction.¹ Among the secretory products of macrophages and foam cells are O₂^- and H₂O₂ (reviewed in Reference 14¹⁴ ), which, in the presence of transition metal ions, may initiate peroxidation of cholesteryl esters and phospholipids in LDL particles; this process results in the nonenzymatic chemical modification of the apoB100 moiety of LDL,¹⁵ a characteristic feature of oxLDL.

PAF, which is synthesized by major proinflammatory cells such as monocytes, macrophages, neutrophils, platelets, and endothelial cells,¹⁶ has been localized in human atherosclerotic plaques.¹⁷ Moreover, we have recently shown in vitro that human monocyte–derived macrophages and macrophage-derived foam cells represent a potential source of PAF in the arterial intima.³ In this context, it is relevant that oxidative modification of LDL may be stimulated by PAF as a consequence of its potent activation of the production of active oxygen species.¹⁸

Despite the capacity of macrophages to produce PAF, it is indeterminate as to whether these cells express the membrane receptor specific for this ether-phospholipid agonist and equally whether the level of receptor expression may be regulated by native or modified LDL. The goal of our study was therefore to evaluate PAF receptor expression and activity in human monocyte–derived macrophages and its potential modulation by oxLDL; in addition, we investigated the role of cAMP in receptor regulation. Our data reveal that atherogenic oxLDLs downregulate expression of PAF receptor mRNA and PAF receptor expression in human monocyte–derived macrophages and suggest that a mechanism implicating this cyclic nucleotide is involved in signal transduction. These studies reinforce the hypothesis that downregulation of the PAF receptor is intimately linked to loss of an acute inflammatory phenotype in macrophages exposed to oxLDL.

	Methods

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Materials
Restriction enzymes, plasmids, and molecular size markers for DNA (Promega) were used according to the manufacturer's specifications. The Megaprime random-primer kit was supplied by Amersham International. "RNA plus," phenol, and oligonucleotides were from Bioprobe Systems. DynaZyme thermostable DNA polymerase was obtained from Finnzymes OY. PAF C18:0 (1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine) and carbamyl PAF were supplied by France Biochem. The labeled nucleotides [³²P]dATP (3000 Ci/mmol), [³²P]UTP (800 Ci/mmol), and [³H]octadecyl-9,10-PAF (141.6 Ci/mmol) were purchased from DuPont de Nemours Division NEN. Cell culture media were purchased from BioWhittaker and Superscript Plus M-MLV reverse transcriptase and RNA ladder from GIBCO-BRL. Nutridoma HU medium was supplied by Boehringer Mannheim and pooled human serum (100 to 150 donors) by ATGC. Forskolin, dideoxyforskolin, dibutyryl cAMP, and all other chemical reagents were obtained from Sigma Chemical Co. The following antibodies were from Dako: anti-CD68 (clone KP1), anti-CD14 (clone TÜK4), anti-CD3 (clone T3-4B5), and goat anti-mouse FITC-conjugated IgG. The chromogenic Limulus amebocyte lysate assay for lipopolysaccharide was purchased from Biogenic. Competitive PCR experiments were performed in the presence of DNA-Mimic from Clontech Laboratories. The assay kits for LDH and BCA protein determination were purchased from Boehringer and Pierce, respectively.

Isolation and Culture of Human Monocyte– Derived Macrophages
Mononuclear cells were isolated from the blood of healthy, normolipidemic donors (thrombopheresis residues) as described.¹⁹ Cells were plated at a density of 1.5x10⁶ per well into 15-mm plastic culture dishes in RPMI medium containing gentamicin (40 µg/mL), glutamine (0.05%), and human serum (10%) for receptor binding assays and at 3x10⁶ per well into 35-mm plastic culture dishes for RNA assays. At day 12 of culture, monocyte-derived macrophages (denoted as macrophages) were washed three times with PBS and then incubated for defined times with LDL or oxLDL (50 to 125 µg protein per milliliter) in the same medium (500 µL), except that human serum was replaced by 1% Nutridoma. All cell cultures and incubations were carried out in a humidified 37°C incubator (5% CO₂/95% air atmosphere). Cell viability was measured by trypan blue exclusion and the release of LDH into the medium.

Characterization of Human Monocyte– Derived Macrophages
Mononuclear cells were characterized by application of a panel of specific antibodies and visualized by indirect immunostaining. These cells were positive for CD14 and CD68 markers of monocytes and CD3 antigen, which is characteristic of T cells. At day 12 of culture, monocytes had differentiated into macrophages and were free of lymphocytes. Indeed, adherent cells were all CD68-positive but negative for CD3.

Purification and Chemical Modification of Lipoproteins
LDLs in the density interval 1.025 to 1.050 g/mL were isolated from normolipidemic human plasma by sequential ultracentrifugation and exhaustively dialyzed at 4°C against 0.01 mol/L degassed PBS (pH 7.4) containing 3 mmol/L EDTA (PBS-EDTA). The purity of each LDL preparation was evaluated as described²⁰ ; protein content was determined with the use of the BCA assay kit. Before chemical modification, LDLs were dialyzed against PBS (pH 7.4) to remove EDTA. Copper-oxidized LDLs were prepared under sterile conditions by incubating 500 µg LDL protein per milliliter in PBS containing 2.5 µmol/L CuCl₂ for 48 hours at 37°C. At the end of the incubation period, oxLDL was extensively dialyzed at 4°C, first against PBS at pH 7.4 and then against RPMI 1640, and subsequently filtered through a 0.22-µm filter (Millipore). The time course of copper-induced oxidation of LDL was deduced from the spectrophotometric measurement of conjugated diene formation at 234 nm. The net electrical charge on both native and oxLDL at pH 8.6 was estimated by electrophoresis in agarose gel.²¹ The electrophoretic mobility was expressed as the REM of oxLDL relative to native LDL. The degree of lipid oxidation was estimated on the basis of the contents of thiobarbituric acid– reactive substances²² and lipid hydroperoxides.²³ The endotoxin content of oxLDL was measured before its addition to the cell culture medium by use of the chromogenic Limulus amebocyte lysate assay; only those preparations with <50 pg endotoxin per 100 µg oxLDL protein were used.

Binding Assays of [³H]PAF to Macrophages
Before the binding assays, cell monolayers were washed three times with RPMI 1640 (devoid of phenol red) containing 0.5% BSA (fatty acid free). Binding assays were performed directly in the culture dishes at 20°C for 30 minutes in a final volume of 300 µL with 4.2 mmol/L HEPES-saline buffer containing 1.3 mmol/L CaCl₂, 1 mmol/L MgCl₂, and 0.25% BSA at pH 7.4. We used a range of [³H]PAF concentrations from 0.25 to 2 nmol/L. In some experiments, the binding assays were performed at a unique [³H]PAF concentration of 1 nmol/L. Specific binding was determined as the total radioactivity bound minus the radioactivity bound in the presence of 1 µmol/L unlabeled PAF or PAF antagonist WEB 2086 (nonspecific binding). The binding reaction was terminated by eight rinses with cold HEPES-saline buffer at pH 7.4 (CaCl₂, MgCl₂, and BSA omitted). Adherent cells were lysed in 0.5 mL of 0.1N NaOH and then counted for radioactivity. Binding parameters were determined from equilibrium binding studies by linear transformation. The average number of PAF binding sites per cell and the dissociation constant were calculated by Scatchard analysis. The degree of [³H]PAF degradation in the binding assay was estimated as follows. Incubations were terminated by extraction of [³H]PAF with four volumes of absolute ethanol²⁴ ; the extracts were then brought to dryness under an N₂ stream and analyzed by liquid chromatography on a Microporasil column (Millipore) eluted with chloroform/methanol/water (1:1:1, vol/vol/vol).²⁵

RNA Isolation
Total RNA was isolated from adherent macrophages in six-well culture dishes with RNA Plus according to the protocol of the manufacturer. RNA concentrations were spectrophotometrically determined at 260 nm.

First-Strand cDNA Synthesis and RT-PCR
First-strand cDNA synthesis was performed with 5 or 10 µg of total RNA. Immediately before use, the RNA was heated for 5 minutes at 70°C with 2 U RNAsin, 2 µg oligo-dT, and 1 µg antisense oligonucleotide PAF-3 (5'-ACTTTTCGGTGAGGTGCTTG-3'; nucleotides 1169 to 1188) in aqueous solution in a total volume of 30 µL. After denaturation, RT was performed at 37°C for 1 hour in a total volume of 50 µL containing 1x reverse transcriptase buffer, 0.5 mmol/L dNTP, 10 mmol/L DTT, and 500 U SuperScript plus M-MLV reverse transcriptase. Detection and quantification of PAF receptor mRNA were performed by RT-PCR in the presence of two specific oligonucleotides, PAF-1 (5'-CCGATACACTCTCTTCCCGA-3'; nucleotides 151 to 170) and PAF-2 (5'-ACAGTTGGTGCTAAGGAGGC-3'; nucleotides 970 to 951). The numbering is in accordance with the cDNA sequence of the human leukocyte PAF receptor (GenBank HUMPAFRE). The incubation volume was adjusted to 50 µL by adding master mix components to the first-strand cDNA dilutions and overlaid with one drop of mineral oil; final concentrations were 1x DynaZyme buffer, 0.2 mmol/L dNTP, 100 ng oligonucleotide of each upstream and downstream primer, and 1 U DynaZyme DNA polymerase. Incubations were performed in a Techne Thermal Cycler, starting at 94°C for 5 minutes followed by 30 cycles of 1 minute, 15 seconds at 94°C; 1 minute, 15 seconds at 60°C; and 2 minutes, 15 seconds at 72°C successively; these cycles were followed by 1 cycle for 7 minutes at 72°C before storage at 15°C. PCR products were analyzed by fractionation of 10-µL aliquots on a 2% agarose/TAE gel. Control samples analyzed without reverse transcriptase were free of genomic DNA.

Quantification of Actin by Competitive PCR
Competitive PCR experiments were performed in the presence of DNA-Mimic from Clontech Laboratories (0.01 to 1 amol per assay), 1 µL of first-strand cDNA diluted 100-fold, and [³²P]dATP with actin-specific oligonucleotides. The PCR products (actin, 838 bp; mimic, 619 bp) were analyzed by separating 10-µL PCR aliquots on a 2% agarose gel and compared on the basis of intensities of ethidium bromide–stained bands or by counting ³²P-labeled bands after slicing the gel. The first-strand cDNA was then diluted to obtain an equivalent amount of actin before amplification of PAF receptor mRNA. Similar competitive experiments were performed with GAPDH.

Northern Blot Analysis
RNA samples (20 µg) were fractionated by electrophoresis on 1% agarose/formaldehyde gels and transferred to Hybond N⁺ membranes. An antisense riboprobe (Promega System II) was transcribed by T7 polymerase from its promoter in a linearized BlueScript construct (BamHI) corresponding to 2 kb of human PAF receptor cDNA (a gift from Dr Shimizu, Tokyo, Japan). RNA blots were prehybridized for 5 to 17 hours and hybridized with the riboprobe (10⁶ cpm/mL) for 20 hours at 60°C in 50% formamide, 5x SSPE, 5% SDS, and 100 µg/mL salmon sperm DNA. The blot was washed at 70°C for 30 minutes in the presence of 1x SSPE with 0.5% SDS and then with 0.1x SSPE containing 0.5% SDS and exposed to x-ray film (Amersham MP) for 1 to 7 days. The electrophoretic mobilities of mRNAs were determined by comparison with an RNA ladder. All blots were reprobed with an actin cDNA probe. The intensities of each band were evaluated by videoscanning with an Imager 220V (Appligene).

	Results

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PAF Receptor Expression in Human Monocyte–Derived Macrophages
Human monocyte–derived macrophages cultured for 12 days with human serum were exclusively CD68-positive and CD3-negative, as assessed by indirect immunofluorescence staining with specific antibodies. In addition, these cells typically expressed mRNA for both scavenger receptors²⁶ and lipoprotein lipase (D.S. et al, unpublished data, 1995), such expression being characteristic of human macrophages.

The presence of mRNA for the PAF receptor in macrophages was initially evaluated by Northern blot analysis followed by RT-PCR. Northern blots hybridized with a cRNA probe revealed the presence of three distinct size species of PAF receptor mRNA corresponding to 3.3, 2.8, and 1.8 kb (Fig 1), as previously reported in rat brain.²⁷ The presence of different transcripts corresponding to the PAF receptor on Northern blots was further confirmed by RT-PCR, as shown in Fig 2. By nonquantitative PCR, expression of two different 5' sequence regions of PAF receptor cDNA was amplified with specific primers⁸ in an RT-PCR assay performed with three sets of oligonucleotides: (1) the common oligonucleotides (PAF-1/PAF-2) for both PAF-1 and PAF-2 promoters; (2) the L1/C1 oligonucleotides corresponding to promoter 1 (the leukocyte type); and (3) the H1/C1 oligonucleotides for promoter 2 (the heart or spleen type), as shown in Fig 2 (lower panel). mRNA extracts from human brain, testis, monocytes, and macrophages were probed for the presence of transcripts corresponding to promoters 1 and 2. These studies demonstrated that not only do human brain and testis express abundant transcripts corresponding to promoter 1 of the PAF receptor gene but so also do human monocytes and adherent human macrophages (Fig 2, upper panel). By contrast, human brain and adherent macrophages expressed only small amounts of mRNA transcripts for promoter 2 of the PAF receptor.

View larger version (29K): [in this window] [in a new window]   Figure 1. Northern blot analysis of mRNA extract from monocyte-derived macrophages hybridized with human PAF receptor cRNA. Total mRNA extract (20 µg) from untreated macrophages at 12 days of culture was hybridized with a 2-kb antisense cRNA corresponding to the human PAF receptor as described in "Methods." RNA markers are shown at left. The size of each PAF receptor mRNA species is indicated at the right.

View larger version (39K): [in this window] [in a new window]   Figure 2. PCR analysis of tissue specificity of the expression of two forms of promoter transcripts for the human PAF receptor gene. Amplification products were obtained from human brain (HB), testis (HT), monocytes (MO), and adherent macrophages (MA, MA/oxLDL) by using primer pairs specific for the respective PAF receptor region. Primer pairs include the following: the common region PAF-1/PAF-2 (819 bp), promoter 1 L1/C1 (191 bp), and promoter 2 H1/C1 (252 bp). PCR was performed as described in "Methods" and each product was examined by electrophoretic separation on 2% agarose gels. After separation, DNA transcripts were detected by ethidium bromide staining. Oligonucleotide primers specific for promoters 1 and 2 were taken from Mutoh et al8 ; oligonucleotide primers from Clontech were used as controls for actin (838 bp).

We next examined the binding of [³H]PAF to its receptor on adherent human macrophages. Fig 3 shows that the specific binding of PAF to macrophages was concentration dependent and attained saturation. Scatchard analysis revealed a K_d of 2.5±0.9 nmol/L and a B_max value of 8.7±1.6 fmol/10⁶ cells, corresponding to 5270±990 binding sites per cell. The variability observed in these experiments arose from preparation of human monocytes/macrophages from the blood of different donors. The specific PAF antagonist WEB 2086 displaced the specific binding of [³H]PAF to control macrophages in a manner similar to unlabeled PAF, although the concentration used (10 µmol/L) was 10-fold higher than that of unlabeled PAF (Table). The absence of significant metabolism of [³H]PAF under our experimental conditions was examined by liquid chromatography: >95% of the initial [³H]PAF added was recovered as intact PAF in three independent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 3. Scatchard analysis of [3H]PAF binding to control adherent human macrophages and cells pretreated with oxLDL. Typical Scatchard transformations of [3H]PAF binding data determined in human macrophages treated with oxLDL (100 µg protein per milliliter) for 48 hours () and control data for cells incubated without oxLDL () are shown. Bmax values were 19 fmol/mg protein for control macrophages and 7 fmol/mg protein for oxLDL-treated cells (P<.005, n=3). Kd values for [3H]PAF binding were determined as 2.1±0.9 nmol/L for control macrophages and 1.6±0.1 nmol/L for oxLDL-treated cells (P<.07, n=3) and were from four separate experiments in which each point was assayed in triplicate. Data are mean±SD.

View this table: [in this window] [in a new window]   Table 1. Effect of OxLDL on Binding of [3H]PAF to Human Monocyte–Derived Macrophages

Effect of oxLDL on Expression of the PAF Receptor in Human Macrophages
We subsequently evaluated the effect of copper-oxidized LDL on the expression of PAF receptor mRNA and binding of PAF to its receptor in macrophages. The preparations of extensively oxLDL used in these experiments were characterized by classic criteria: (1) electrophoretic mobility on agarose gel, reflecting the elevated net negative charge of oxLDL compared with that of native LDL (REM, 4.8±0.2; n=3); (2) generation of aldehydes as estimated by thiobarbituric acid–reactive substances content (0.8±0.7 and 39.5±3.3 nmol/mg protein, respectively, for native and oxLDL [P<.0001, n=3]); and (3) lipid hydroperoxide content in native and oxLDL (10.8±3.4 and 293.4±86.2 nmol/mg LDL protein, respectively [P<.003, n=3]).

Macrophages were incubated at 37°C with oxLDL for increasing times up to 48 hours. After 18 hours of incubation with oxLDL (100 µg oxLDL protein per milliliter), we observed a significant increase in cellular cholesteryl ester content (12.7±1.6 µg/µg DNA in control macrophages and 25.3±1.3 µg/µg DNA in oxLDL-treated cells, respectively; P<.003, n=3), suggesting that such macrophages had undergone lipid loading. The viability of monocyte/macrophage cultures was 95% by the trypan blue exclusion test. In addition, we detected a 15% release of LDH after the 48-hour treatment with oxLDL (100 µg protein per milliliter); the latter value is slightly higher than that typical of control cultures lacking oxLDL (10% release of LDH at 48 hours).

The effect of oxLDL on the level of PAF receptor mRNA expression in macrophages was then assessed. Macrophages were first treated for 6 hours with oxLDL at 62.5 or 125 µg protein per milliliter. Owing to the paucity of PAF receptor mRNA in macrophages, we developed a highly sensitive RT-PCR method that was first calibrated with a mimic cDNA for actin and GAPDH (see "Methods"). RT-PCR amplification was linear under our experimental conditions (Fig 4A). As shown in Fig 4B, the abundance of the PAF receptor PCR product decreased by 35% after treatment with 62.5 µg protein per milliliter oxLDL and by 75% after treatment with 125 µg protein per milliliter oxLDL. In the same experiment, the abundance of RT-PCR products corresponding to scavenger receptor type 1 and 2 mRNAs was not modified by oxLDL (Fig 4C). When macrophages were treated with or without native LDL (100 µg protein per milliliter), we could not detect any modification of PAF receptor mRNA expression after incubation for up to 18 hours (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 4. Effect of oxLDL on mRNA levels for PAF and scavenger receptors in adherent human macrophages. Adherent macrophages (A) were treated with oxLDL at 62.5 (B) or 125 (C) µg protein per milliliter for 6 hours in culture. Total RNA was extracted, transcribed, and subjected to PCR with specific oligonucleotides for actin, PAF receptor (), and scavenger receptor types I () and II () as described in "Methods." A, Upper panel: competitive PCR experiments were performed with DNA-Mimic from Clontech Laboratories (0.01-1 amol per assay) with 2 µL of first-strand cDNA diluted 100-fold with oligonucleotides specific to actin as described in "Methods." Lower panel: different amounts of cDNA were assayed for PCR linearity after 30 cycles as described in "Methods." B, After normalization with actin, synthesis of cDNA specific to each receptor was expressed as a percentage of the initial amount in control untreated cells. Values are mean±SD of 3 separate experiments. C, Characterization of scavenger receptor types I and II by ethidium bromide.

Fig 5 shows the time course of changes in PAF receptor mRNA level in macrophages treated with oxLDL (100 µg protein per milliliter), as determined by RT-PCR. The abundance of PAF receptor mRNA diminished by 60% after 1 hour of incubation with oxLDL; at 6 hours, maximal inhibition of 80% was observed and remained at this low level for 48 hours. Because the response of PAF receptor mRNA expression to macrophage treatment with oxLDL was immediate, it suggested to us that modulation of mRNA abundance was independent of protein synthesis and might therefore be due to increased degradation of either PAF receptor mRNA or its posttranscriptional processing.

View larger version (19K): [in this window] [in a new window]   Figure 5. Time course of effect of oxLDL on PAF receptor binding and mRNA expression in human macrophages. Adherent macrophages were treated with 100 µg oxLDL protein per milliliter for 1-38 hours at 37°C. At each time, total mRNA was extracted and binding assayed as described in "Methods." Total mRNA was further transcribed and subjected to PCR with oligonucleotides specific for actin or the PAF receptor. After normalization with actin, PAF receptor cDNA synthesis was expressed as a percentage of the initial amount (). Specific binding of PAF to monocyte-derived macrophages is expressed in disintegrations per minute per well (). Values are mean±SD of 3 separate experiments, each performed in triplicate.

We next assessed the expression of type 1 and 2 promoters of the PAF receptor after macrophage treatment with oxLDL (100 µg protein per milliliter) for 6 hours. These experiments revealed that the PCR product corresponding to the amplified sequence specific to promoter 1 decreased in a manner similar to that corresponding to the common region of mRNA. Expression of promoter 2, which was present at low levels in control macrophages, was not detected in cells treated with oxLDL.

Subsequently, we evaluated the binding of [³H]PAF to its receptor in monocyte-derived macrophages treated with oxLDL. After a 48-hour incubation with oxLDL at 100 µg protein per milliliter, the K_d for [³H]PAF binding was not significantly reduced (1.5±0.2 nmol/L; P<.07, n=3) relative to control cells. In contrast, the number of high-affinity sites for PAF per cell was reduced to about half of that in control cells (2500±330 versus 5268±985 sites per cell, respectively; P<.005, n=3). B_max was also reduced in macrophages treated with oxLDL (4.2±0.6 versus 8.7±1.6 fmol/10⁶ cell, Fig 3). Evaluation of the time course of changes in human PAF receptor activity was performed with 100 µg/mL oxLDL for periods as long as 48 hours; these studies revealed that the number of binding sites decreased by >30% after a 3-hour incubation with oxLDL and remained low for at least 2 days (Fig 5). Displacement of [³H]PAF binding to PAF receptors on oxLDL-treated macrophages was achieved with either the PAF antagonist WEB 2086 (10 µmol/L) or 1 µmol/L unlabeled PAF (Table).

The concomitant decrease in both PAF binding and PAF receptor mRNA level induced by treatment with oxLDL (100 µg/mL) for 6 hours was significantly reversed after the cells were washed and cultured in RPMI medium for 18 hours (P<.005, n=3) (Fig 6). Thus, a reduction in receptor number may be due to a decrease in the level of receptor expression, an increase in the rate of protein turnover, or a combination of both.

View larger version (52K): [in this window] [in a new window]   Figure 6. Effect of oxLDL and various pharmacological treatments on [3H]PAF binding to monocyte-derived macrophages (12-day culture) and expression of PAF receptor mRNA. Specific binding of [3H]PAF (in disintegrations per minute) and amount of RT-PCR product of PAF receptor mRNA (as a percentage of control values) were measured as described in "Methods." Pretreatment of macrophages (6 hours at 37°C) was as follows: 100 µg protein per milliliter oxLDL, 1 mmol/L dibutyryl cAMP, 3 µmol/L forskolin, 30 µmol/L dideoxyforskolin, and 100 nmol/L carbamyl PAF. The assay to demonstrate reversibility of the effect of oxLDL was performed on macrophages treated for 6 hours with oxLDL (100 µg protein per milliliter) followed by two washes with PBS and subsequent culture in fresh medium for 18 hours.

Comparison of the Effect of oxLDL and cAMP or Carbamyl PAF on PAF Receptor Levels in Human Macrophages
Because PAF receptor mRNA levels are downregulated in monocytes by increases in cAMP level,¹⁰ we therefore asked whether the mechanism of oxLDL-induced inhibition of mRNA expression in macrophages could be mediated by changes in intracellular cAMP level. Thus, both forskolin (3 and 30 µmol/L), a powerful adenylyl cyclase activator, and the cell-permeable cAMP analogue dibutyryl cAMP (1 mmol/L) were found to reduce not only PAF binding (P<.005, n=3) but also mRNA level in a manner similar to that of oxLDL (Fig 6). The effect of dideoxyforskolin, a forskolin analogue that acts independently of adenylyl cyclase, was also tested. Indeed, both PAF binding and expression of PAF receptor mRNA (P<.05, n=3) were reduced by high concentrations of dideoxyforskolin (30 µmol/L), although the degree of inhibition never exceeded that induced by forskolin. Such inhibition was observed with or without oxLDL (data not shown). Conversely, elevations in cAMP level or macrophage treatment with oxLDL (100 µg/mL) induced a 50% reduction in cellular binding sites for [³H]PAF and a similar degree of inhibition of human PAF receptor gene expression (Fig 6).

Carbamyl PAF has been reported to downregulate PAF receptor gene expression in U937 cells.²⁸ We therefore compared the effect observed with oxLDL to that resulting from ligand binding to the PAF receptor. Carbamyl PAF (100 nmol/L), a nonhydrolyzable analogue of biologically active PAF, reduced PAF binding and the abundance of PAF receptor mRNA in macrophages in the same manner as that observed with oxLDL (Fig 6), thereby suggesting that oxLDL can mimic the effect of PAF.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Using RT-PCR and Northern blot analysis, we have demonstrated for the first time that human monocyte–derived macrophages constitutively express PAF receptor mRNA transcripts as well as specific binding sites for this mediator. By RT-PCR, such macrophages were shown to express at least two transcripts, ie, the leukocyte type (1) and the heart type (2).⁸ Expression of transcript type 2 has not yet been described in circulating cells; indeed, it was believed to be characteristic of organs such as the heart, spleen, and kidney.⁸ Furthermore, when macrophages were treated with atherogenic copper-oxidized LDL, a marked decrease in PAF receptor mRNA (transcript 1) expression occurred as early as 1 hour after treatment. Under the same experimental conditions, expression of transcript 2 was abolished.

Expression of the PAF receptor at the surface of macrophages was characterized by using [³H]PAF as the ligand. A single class of PAF receptor with high affinity for PAF (K_d=2.1 nmol/L) was revealed, as has previously been shown in murine macrophages.^{29 30} The binding assay revealed that macrophage treatment with oxLDL was accompanied by a marked decrease (50%) in the number of binding sites at the cell surface without any affect on binding affinity. This reduction was detected after a 6-hour treatment with oxLDL (100 µg protein per milliliter) and remained low for at least 48 hours. In a previous study with undifferentiated U937 cells incubated with native LDL, increases in both PAF binding and PAF acetylhydrolase activity were observed.³¹ However, this undifferentiated monocytic cell line displays several differences when compared with human monocytes and includes the capacity to produce acetylhydrolase. These two cellular models are therefore not strictly comparable.

Intracellular cAMP levels regulate expression of numerous receptors. Thivierge et al¹⁰ have recently shown that PAF receptors in human monocytes are downregulated by pharmacological treatments that increase cAMP levels, such as prostaglandin E₂, cholera toxin, or forskolin. In our human macrophage model, forskolin decreased both mRNA expression and PAF receptor binding to an extent similar to that seen with oxLDL treatment. Forskolin exerts multiple effects on target cells, including increases in adenylyl cyclase activities and glucose transport, whereas an analogue of forskolin, ie, dideoxyforskolin, acts independently of adenylyl cyclases. In our experience, dideoxyforskolin, when used at high concentrations, only slightly decreased mRNA expression as well as [³H]PAF binding to macrophages. Thus, the actions of forskolin are probably closely linked to increases in cAMP via the adenylyl cyclase pathway. This observation was further reinforced by experiments with a nonhydrolyzable analogue of cAMP, dibutyryl cAMP.

OxLDL interacts with human macrophages via several mechanisms, including those of the scavenger,¹¹ CD 36,¹² and Fc¹³ receptors; such interaction leads to internalization and cellular degradation of the bound ligand. In addition, oxidized phospholipid components of oxLDL are analogous in structure to PAF, as originally reported by Heery et al.³² Such oxidized phospholipids may therefore interact with the PAF receptor. Indeed, carbamyl PAF, a nonhydrolyzable analogue of PAF, and oxLDL each induced similar decreases in both mRNA and the binding capacity of the PAF receptor. In earlier studies, Chau et al²⁸ reported that expression of the PAF receptor was downregulated by carbamyl PAF in U937 monocyte-like cells. In contrast and as shown herein, native LDL, which contained low levels of lipid hydroperoxides comparable to those reported earlier,³³ had no effect on PAF receptor mRNA level and binding capacity. Lehr et al³⁴ demonstrated that administration of oxLDL to hamsters promoted leukocyte adhesion to the endothelium of venules and arterioles. Such adhesion was inhibited by the PAF antagonist WEB 2170, suggesting that the PAF receptor was directly implicated in such cellular interaction.

PAF may play a pivotal role in atherogenesis. Indeed, phagocytosis-stimulated human monocytes²⁴ and macrophages as well as cholesterol-loaded foam cells transiently produce elevated amounts of PAF,³ which may in turn activate numerous cells in the atheromatous plaque. Thus, PAF activates formation of active oxygen species¹⁸ and elastase release from human macrophages¹⁹ and induces upregulation of the synthesis of growth factors implicated in smooth muscle cell proliferation³⁵ and tumor necrosis factor-–induced angiogenesis.³⁶ In addition, recent studies have shown that PAF plays a major role in the lymphocyte-mediated expression of tissue factor by endothelial cells,³⁷ thereby suggesting that PAF participates in thrombus formation.

PAF-induced signals are attenuated by repetitive or long-standing applications of the agonist, a process frequently referred to as homologous desensitization; such effects are probably due to posttranscriptional phosphorylation of the cytoplasmic tail of the PAF receptor.³⁸ The activity of the PAF receptor may thus be locally diminished at sites of PAF accumulation. In our experiments, we observed a profound decrease not only in PAF binding but also in levels of its corresponding mRNA. Such substantial decreases could not be attributed to homologous desensitization. OxLDL has been shown to be cytotoxic in some models³⁹ ; in our experience, however, the viability of macrophages after 48-hour treatment with oxLDL (125 µg protein per milliliter) as assessed by trypan blue exclusion and LDH release was not affected. Furthermore, the effect of oxLDL on both mRNA expression and PAF receptor binding was reversed by washing the cells, followed by overnight culture in fresh medium. In addition expression of mRNA for scavenger receptors 1 and 2 was not affected by oxLDL.

PAF receptor promoter 1 possesses recognition sites for nuclear factor-B,^{8 40} which may be regulated by oxLDL. Indeed, oxLDL has been reported to suppress lipolysaccharide-induced activation of nuclear factor-B in murine peritoneal macrophages via a pertussis toxin–sensitive signaling route.⁴¹ Furthermore, in endothelial cells minimally modified LDL has been shown to induce inflammatory responses mediated by elevations in cAMP level. This increase was shown to be dependent on Gs or Gi proteins via the adenylyl cyclase system. Activation of the nuclear factor-B transcription factor in this process is thus established⁴² ; equally, PAF stimulates B binding activity in human monocytes through a G protein–coupled pathway.³⁵

The decrease in PAF receptor expression induced by oxLDL in our human macrophage system is relevant to the motility of macrophages and foam cells in the atherosclerotic lesion. Because PAF is chemotactic for leukocytes, the reduction in PAF receptor number at the surface of macrophage/foam cells reinforces earlier findings showing that the motility of foam cells in human atherosclerotic plaques is impaired.⁴³ Indeed, such cells are found exclusively in defined focal areas of atherosclerotic lesions, where they undergo necrosis and contribute to formation of the lipid core of the plaque. In contrast, macrophages that fully express PAF receptors may preserve their mobility and capacity to migrate. Equally, the ability of oxLDL to suppress gene expression is well established in monocytes. Indeed, several inducible inflammatory mediators, such as tumor necrosis factor-,⁴⁴ interferon-,⁴⁵ and interleukin-2,⁴⁶ are negatively regulated in monocytes by oxLDL but not by native or minimally modified LDL. As hypothesized by Hamilton et al,⁴⁶ suppression of an acute inflammatory response may be part of a physiological process allowing development of a state of chronic, low-level inflammation. Such chronic inflammation is consistent with the prolonged period required for the conversion of fatty streaks to more advanced atheromatous plaques. These findings lead us to propose that PAF and oxidized phospholipids interact with the PAF receptor of macrophages and foam cells to contribute to the initiation and progression of atheromatous plaques.

	Selected Abbreviations and Acronyms

 

oxLDL = oxidized LDL PAF = platelet-activating factor PCR = polymerase chain reaction REM = relative electrophoretic mobility RT = reverse transcription

	Acknowledgments

 
This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and partially supported by ARCOL (Comité Français de Coordination des Recherches sur l'Athéroslérose et le Cholestérol) and MRT No. 25C1A036A (French Ministry of Research and Technology, Actions Concertées-Sciences du Vivant). We are most grateful to Drs M. Rola-Pleszczynski and J. Stankova for stimulating discussion and Dr. P. Lesnik for advice in preparation of cell cultures. We are deeply indebted to C. Debets-Albertini (Centre Départemental de Transfusion Sanguine, Créteil, France) for the generous gift of thrombopheresis residues and European Community Concerted Action No. PL-931790.

Received May 21, 1996; accepted August 22, 1996.

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